We propose to elucidate mechanisms of development and regeneration in the inner ear in the zebrafish, Danio rerio. The inner ear arises form a simple thickening on the surface of the embryo termed the otic placode, which subsequently forms the otic vesicle where sensory epithelia and neurons of the ear arise. We have shown that each of these processes requires the cell signaling molecule Fgf. Additionally, various transcription factors belonging to the Pax2/5/8 and Sox2/3 families modify the response to Fgf to help establish different kinds of cell types. In three specific aims we will investigate how thee genes work during development, as well as during regeneration to repair damage that might otherwise cause deafness. 1) We will study how the related factors Sox2 and Sox3 regulate early development of the inner ear. We hypothesize that they initially work together to establish the otic placode. Soon thereafter, Sox2 promotes development of sensory epithelia and Sox3 promotes development of neurons that transmit signals from the ear to the brain. These studies will rely on transgenic lines in which Sox2 or Sox3, or other factors of interest, can be overexpressed by simply incubating embryos at elevated temperatures. 2) We will study how Fgf, Pax2/5 and Sox2 function in the maintenance and regeneration of sensory hair cells. These factors are known to regulate initial development, but we will now use a new technology (photo-MOs) that permit analysis of later functions. For example, we will block gene function after hair cells have formed to determine whether the gene is needed for hair cell survival; and after killing hair cells with a laser, we will determine whether these genes are necessary for regeneration. 3) We will investigate the role of the transcription factor Tfap2a in formation of neurons. Tfap2a has not previously been studied in the inner ear in any species, but we have discovered it has a profound stimulatory effect on neuron production in zebra fish. We will investigate how Tfap2a works, and how it interacts with the other factors known to promote neuron formation, such as Fgf and Sox3. Together, these studies will provide fundamental insights into mechanisms of otic placode induction and sensory/neural development. Because developmental mechanisms are broadly conserved, studying how these genes work in zebra fish could suggest candidates for gene therapy to restore hearing in mammals.